Project Summary/Abstract Kaposi's sarcoma herpes virus (KSHV) is an oncovirus that causes several cancers including Kaposi's sarcoma (KS). It is estimated that 3 to 5% of U.S. blood donors have been infected with KSHV, yet in some high-risk populations such as homosexual men, prevalence is as high as 65%. In spite of HAART, KS remains the second most common AIDS- associated malignancy. A third of AIDS-KS cases now arise in individuals with relatively high CD4+ cell counts and low HIV-1 viral load. A recent study has observed a continuing high prevalence and increased acquisition of KSHV in HIV- 1+ infected persons on HAART. The data clearly indicate that KS is still occurring in HIV-infected individuals, even in the context of successful HAART. In immunosuppressed populations, approximately 1 in 200 transplant patients will develop KS. The prevalence of KSHV is responsible for the continuing incidence of KS and its associated reduction in life expectancy of HIV+ individuals. Despite its strong disease association, particularly in the HIV+ and immunosuppressed populations, there is no FDA approved clinical diagnostic test for KSHV and consequently a significant unmet need remains for a simple, cost-effective commercially viable KSHV diagnostic kit that would be an essential component of any effort to control virus transmission and monitor serostatus of those already infected. The ability to gauge the KSHV serostatus of an HIV+ individual would help clinicians make appropriate interventional choices to prevent KS onset. The overall goal of this project is to develop a serological multi-antigen assay for the detection of KSHV antibodies that can be used commercially to diagnose at-risk patients and identify blood/tissue donors with KSHV infection. In prior SBIR Phase I and II work, Epiphany developed a single-well, multi-antigenic KSHV enzyme linked immunosorbent assay (ELISA) called ?4-Combo.? In screening a sera panel containing both KSHV true- positives and normal, low-risk blood donors, 4-Combo was the only assay out of 7 other independent ELISAs to detect all consensus KSHV samples with excellent specificity and sensitivity (estimated >99%, 98%, respectively). For the next stage of the kit development, it is critical to examine 4-Combo's performance against large sera banks of epidemiologically validated KSHV positive and negative controls with relevant confirmatory assays to establish the true effectiveness of the assay. A collaborative consortium that merges Epiphany with the biomedical and clinical research capabilities of the University of Miami (UofM) Miller School of Medicine, the Miami Center for AIDS Research (CFAR) and the labs of Dr. Enrique A. Mesri (CFAR) has been created. Together, Epiphany and Miami CFAR are uniquely equipped to achieve the following Aims. In Aim 1, synthetic, manufacturing, and analytical process development will be implemented followed by pilot production of a set of 4-Combo diagnostic ELISA kits. In Aim 2, these kits will be assessed and optimized by screening retrospective clinical blood samples and serologically characterized plasma/ PMBCs and validated by western blot. In Aim 3, large-scale kit production will commence. Assay stability and validation studies will be performed. A larger panel of HIV+ sera and tissues available to UofM CFAR, its cores and transplant centers, and national repositories will be massively screened to establish sensitivity and specificity. Achieving the goals of this project will establish the clinical and commercial value of 4-Combo ultimately resulting in the first FDA-approved clinical diagnostic assay for KSHV.